Cutana pag mnase

CUT&RUN was performed as previously described (93 (link)). For each sample, around 30 third instar larval CNS were dissected in cold PBS. All experiments were performed in duplicates. Tissues were bound to BioMag Plus Concanavalin A–conjugated magnetic beads (Polysciences Inc.) before blocking and permeabilization in dbe + buffer containing 20 mM Hepes, 150 mM NaCl, 0.1% BSA, 0.5 mM spermidine, 2 mM EDTA, 5% digitonin, and cOmplete EDTA-free protease inhibitor cocktail. The antibodies (table S4) were diluted (1:100) in the same buffer before overnight incubation at 4°C. Samples were washed with dbe + buffer and then incubated for 1 hour at room temperature with Proteins A and G fused to Micrococcal Nuclease produced in E. coli for CUT&RUN Assays (CUTANA pAG MNase) (EpiCypher, #15-1016) diluted in dbe + buffer. After a wash in dbe + buffer, DNA was cleaved in wash + C buffer on ice for 30 min. The cleavage reaction was stopped, and samples were treated with ribonuclease A for 30 min at 37°C. After 2 hours of proteinase K treatment at 50°C, DNA was recovered using Ampure XP beads (Beckman Coulter). Library preparation and sequencing were performed as detailed for ChIP-seq.

CUT&RUN was performed on the embryonic and adult prefrontal cortex as previously described (Skene and Henikoff 2017 (link); Brodie-Kommit et al. 2021 (link)). Three biological replicates were included for each age and genotype. Briefly, E14 mouse embryonic cortex or P60 mouse prefrontal cortex were dissected out, and nuclei were isolated using Nuclei EZ Prep Buffer (Sigma-Aldrich 4432370) and counted on cell cytometer. 300k nuclei were bound to the Concanavalin A-coated beads for each CUT&RUN reaction. Then, each aliquot of bead/nuclei were incubated with a primary antibody, including Rb-MYT1L (0.5 µg, Millipore ABE2915), Rb-H3K4me1 (1 µg, Abcam ab8895), Rb-H3K4me3 (1 µg, Active Motif 39159), Rb-H3K27ac (1 µg, Active Motif 39133), and Rb IgG (1 µg, Jackson ImmunoResearch 011-000-003), at 4°C on the nutator overnight. Next, to bind pAG-MNase fusion protein to the antibodies, beads were incubated with diluted CUTANA pAG-MNase (1:20, EpiCypher 15-1016) on the rotator at 4°C for 1 h. Chromatin digestion was performed at 0°C with the addition of CaCl₂ (100 mM) for 30 min. To digest the RNA and release the cleaved DNA fragments, reactions were incubated with Stop Buffer at 37°C for 30 min in the thermocycler. Magnetic stands were used to bind beads afterwards, and supernants containing DNA fragments were retrieved for sequencing library preparation.

CUT&RUN was performed as described (27 (link)). Briefly, 500 000 cells were washed with Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine and protease inhibitors), bound to Concanavalin A-coated magnetic beads (Bangs Laboratories) and incubated with SOX9 antibody (1:100, AB5535, Millipore) diluted in wash buffer containing 0.05% digitonin (Dig-Wash) overnight at 4°C. Cells were washed and incubated with Guinea Pig anti-Rabbit secondary antibody (1:100, Novus Biologicals) diluted in Dig-Wash for 1 h at RT, washed again and incubated with CUTANA™ pAG-MNase (Epicypher) for 10 min at RT. Slurry was washed, placed on ice and incubated with Dig-Wash containing 2 mM CaCl₂ for 30 min to activate digestion. Stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% Digitonin, 0.05 mg/ml glycogen, 5 μg/ml RNase A) was added, and fragments were released by 30 min incubation at 37°C. DNA was extracted with phenol-chloroform and ethanol precipitation. Libraries were constructed and sequenced. Two biological replicates of SOX9 at 96 h and one replicate of the SOX9 time-course were sequenced.

CUT&RUN coupled with high-throughput DNA sequencing was performed using antibodies targeting SOX4 (Sigma, Catalog: HPA029901, Lot: D116454), SOX11 (Sigma, Catalog: HPA000536, Lot: BG117774), and Cutana pA/G-MNase (Epicypher) according to the manufacturer’s protocol. Briefly, SMS-KCNR cells (5.0 × 105 per reaction) were washed and incubated with activated Concanavalin A beads for 10 min at room temperature. Cells were then resuspended in antibody buffer containing 0.01% digitonin, 1 mL of each antibody was added to individual cell aliquots, and tubes were rotated at 4 °C overnight. The following day, targeted chromatin digestion and release was performed with 2.5 mL Cutana pA/G-MNase and 100 mM CaCl₂. Retrieved genomic DNA was purified with the MinElute PCR purification kit and eluted in 10 mL of buffer EB. Sequencing libraries were prepared with the automated Swift 2S system, followed by 100bp-PE sequencing with Novaseq 6000.

2 × 10⁵ short-term cultured CAFs before or after 30 min of serum stimulation were processed for CUT&RUN^{158 (link)} for macroH2A1 (antibody ab37264, Abcam, lot number GR278020-1, 1 μg per reaction). Cells were permeabilized with 0.0085% digitonin and incubated with antibody. DNA was cleaved with the CUTANA pAG-MNase (Epicypher). Released DNA was processed using a NEBNext Ultra II Library Preparation kit for Illumina (NEB), including a 14-cycle amplification step. Libraries were sequenced and reads were processed as for ATAC–seq.